Bio plex pro human cytokine 27 plex kit

Supernatants harvested from disrupted lung were stored at −80°C and inactivated by exposure to 5 MRAD γ-irradiation on dry ice using a JL Shepherd Model 109–68 Cobalt-60 Research Irradiator (JL Shepherd & Associates, San Fernando, CA). Sterility was confirmed by lack of CFU following 3 weeks of growth on 7H11 agar. Analysis of lung cytokines and chemokines affected by treatment was performed with a human multiplex ELISA (Bio-rad Bio-plex Pro™ human cytokine 27-plex kit) according to the manufacturer's instructions as previously described (Nusbaum et al., 2016 (link)). Results from molecules with cross-reactivity between human detection reagents and mouse cytokines (e.g., VEGF, IL-13) were excluded from the analysis to reduce confounding outcomes. Values that were out of range high were not observed in any of the samples tested. The out of range low values were set to zero and data from tissue analytes where many values were out of range low are not presented. Analytes were quantified by generating a standard curve using validated standards and values were determined by linear regression to the standard curve as described (Nusbaum et al., 2016 (link)).

Supernatants from disrupted lung were harvested during necropsy 5 wk post-M.tb infection and stored frozen at −80 °C. Samples were subsequently γ-irradiated on dry ice using a JL Shepherd Model 109–68 Cobalt-60 Research Irradiator (JL Shepherd & Associates, San Fernando, CA 91340) until 5 MRAD of exposure was reached. A non-diluted test sample was plated on 7H11 agar and incubated at 37 °C for >5 weeks to ensure sterility prior to removing samples from biocontainment. Lung supernatants were used in performance of a multiplex ELISA (Bio-rad Bio-plex Pro™ human cytokine 27-plex kit) according to the manufacturer’s instructions to assess changes in expression of human cytokines: IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, IP-10, FGF basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF. Values for cytokines where cross reactivity between human detection reagents and mouse cytokines could confound data (e.g. VEGF, IL-13) were excluded from the analysis. Levels of each cytokine were quantified by generating a standard curve using the standards provided in the kit. Values were generated by linear regression to the standard curve as recommended by the manufacturer and as described62 (link). Values that were below the extrapolated range (

Short-term cell culture supernatants were thawed and tested (in duplicate, undiluted) using the Bio-Plex Pro™ Human Cytokine 27-plex kit (M500KCAF0Y, Bio-Rad) following the manufacturer’s instructions. Protein standards were provided in the kit, and standard curves were generated with eight standard dilutions (undiluted, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187) using a five-parameter logistic curve fit and 1/y² weighted function. Data were acquired on a MAGPIX instrument (Bio-Rad).